CD20 molecule, with a molecule weight of about 35 kDa, is one of members of MS4A protein family which has four transmembrane domains and contains both their C and N ends within the cell (Cragg, et al, Curr. Dir. Autoimmun, 8: 140-174), CD20, as a membrane protein which expresses on the surface of B lymphocytes, closely relates to Ca.sup.2+conductance in B lymphocytes and regulates the differentiation and proliferation of B lymphocytes. CD20 is exclusively expressed on pre-B lymphocytes, premature B lymphocytes, mature B lymphocytes and activated B lymphocytes, but absent in plasma cells, lymphoid pluripotent stem cell cells and other tissues. The natural ligand of CD20 is still unknown. Because CD20 is highly expressed by more than 90% B-cell Non-Hodgkin's lymphoma, CD20 is an ideal target for antibody therapy of B-cell lymphoma (Blood 63 (6): 1424-1433, 1984). The anti-CD20 monoclonal antibody (mAb) has been proved to have an outstanding in vivo and in vitro effect on B-cell lymphoma (Reff M E et al, Cancer Control 2002; 9:152-66). Upon binding to CD20, anti-CD20 antibody may kill tumor cells through ADCC (Clynes R A et al. Nat Med, 2000; 6:443-6), through. CDC (Harjunpaa A, et al, Scand J Immunol 2000; 51:634-41), and/or direct induction of apoptosis of tumor cell (Apoptosis; Pedersen I M, et al, Blood 2002; 99:1314-9). Because anti-CD20 antibody only kills B cells carrying CD20 surface antigen, it greatly reduces the side effect compared with chemotherapy, thus provides an effective and safe targeting therapy for NHL patients. In recent years, basic research and clinic application of anti-CD20 antibody has been widely investigated. Rituximab, ZEVALIN® (ibritumomab tiuxetan) and BEXXAR® (tositurnornab and iodine I131) have been approved by FDA for treatment of NHL in 1997, 2002 and 2003, respectively. Rituximab is a human-mouse chimeric monoclonal antibody containing variable region derived from murine monoclonal antibody and constant region from human IgG1 heavy and K light chain (Gopal and Press, J Lab Clin Med, 1999; 134:445-450). ZEVALIN® (ibritumomab tiuxetan)is a murine monoclonal antibody conjugated with radionuclide 90.sup.Y. BEXXAR® (tositumomab and iodine I131)is murine monoclonal antibody conjugated with radionuclide .sup.131I. Treatment of refectory NHL with rituximab only achieved a response rate of around 50%; when combined with chemotherapy drug, the response rate can reach up to 90-100%. Because CD20 is also expressed in hairy cell leukemia and B-cell chronic lymphocytic leukemia, more and more indications for anti-CD20 therapy have been applied recently. Rituximab, in combination with chemotherapy, has provided satisfying effect on chronic lymphocytic leukemia (Cheson, et al. 2006, Cancer Immunol. Immunother. 55: 188-196). Due to its efficient therapeutic effect and less side effects, rituximab became one of the bestselling anti-cancer drugs with close to 5 billion sales in 2008.
In addition, B cells also play a central role in pathogenesis of autoimmune disease. Many autoimmune diseases are associated with abnormal activation of B-cell, production of autoantibody and mediation of autoimmune response. Examples include acute idiopathic thrombocytopenic purpura and chronic idiopathic thrombocytopenic purpura, myasthenia gravis, lupus nephritis, lupus erythematosus and rheumatoid arthritis. The most common treatments are corticosteroids and cytotoxic drugs, which can be very toxic. These drugs have adverse effects on the bone marrow, liver and kidney, also suppress the entire immune system and result in serious infection. Therapies directed against B cell have been proved to be effective on the treatment of human autoimmune disease, Rituximab is effective on decreasing B lymphocytes in vivo. Such treatment can eliminate auto-antibodies produced by B cells. Rituximab is furthermore found use in autoimmune diseases, such as rheumatoid arthritis, wherein it has been shown to markedly improve symptoms. Rituximab has been approved by FDA for patients suffered from moderate to advanced rheumatoid arthritis (Summers et al, 2005. Ann. Pharmacother. 39: 2091-2095).
However, the biggest problem of administrating therapeutic antibody is the immune response resulting from the human response to foreign antibody protein, namely anti-antibody reaction (AAR). The murine antibody; working as foreign protein, can stimulate the human body repeatedly and lead to hyper allergic reaction, namely human anti-mouse antibody reaction (HAMA). This leads to the neutralization of murine antibody by anti-antibody, fast clearance of murine antibody or even leads to death caused by severe allergic reaction. In addition, due to the lack of effector function of human antibody (such as ADCC and CDC effect), the therapeutic effect of murine antibody is compromised. Although rituximab is human-mouse chimeric antibody comprising human Fc fragment, it still contains murine antibody sequence in the variable region of both heavy chain and light chain. The murine derived sequence was considered foreign to human system and causes immunogenicity that leads to production of neutralizing antibody and reduces drug efficacy when administered constantly. Especially in the cases of long period treatment with a lower dose, it is more likely to have immunogenicity. Higher immunogenicity of rituximab was exhibited in its treatment of autoimmune disease (such as SLE) than that of NHL (Cantron G. et al, Critical Reviews in Oncology/Hematology, Volume 62).
In order to further decrease the immunogenicity, “CDR graft” became major procedure of humanization of antibody (Riechman et al. Nature, 332:323-327, 1988). Such technique reduces the immunogenicity caused by mouse frame sequence through grafting mouse CDR regions onto framework of human antibody. Currently, this technique has created humanized anti-CD20 antibody 2H7 (Genentech, CN 101418045A), A20 mAb (Immunomedics, Inc., CN 1662557A) and H1286/1373 (VACCINEX), 1K1791 mAb (Osaka University, WO 2009031230 A1) as well as 9D3 mAb (CP Guojian Pharmaceutical Co., Ltd).
Even though the combination of amino acids of these humanized framework and murine CDR still can be recognized by T cells and cause immune response. The recognition of foreign protein by T cell is mainly due to the presentation to T cell receptor by MHC-II expressed on APC surface which binds to protein polypeptide. This further leads to activation of lymphocyte and immune reaction. Therefore, when designing and choosing the humanized framework, the peptide sequence from the framework should have minimum binding with MHC II molecules (HLA-DR), thus minimizes immune response caused by humanized antibody. The humanized antibody of the present invention is based on the sequence of murine 2B8 (Reff, M, E. et al (1994) Blood 83:435-445) and the humanization method described in this application. Through design and selection of the human germline framework sequence that has minimal binding sites with MHC II (HLA-DR), humanized 2B8 molecules were created with much lower immunogenicity. Moreover, using method of molecular docking, CDR sequence can be further modified to maintain or improve antigen recognition and in vivo and/or in vitro anti-tumor activity. These humanized anti-CD20 antibodies also have widely application in the treatment of abnormal activation of B cell and autoimmune diseases.